WO1998036636A2 - Procede de production de cultures cellulaires embryogenes pour la production de bananes transgeniques (de l'espece musa) - Google Patents

Procede de production de cultures cellulaires embryogenes pour la production de bananes transgeniques (de l'espece musa) Download PDF

Info

Publication number
WO1998036636A2
WO1998036636A2 PCT/EP1998/001125 EP9801125W WO9836636A2 WO 1998036636 A2 WO1998036636 A2 WO 1998036636A2 EP 9801125 W EP9801125 W EP 9801125W WO 9836636 A2 WO9836636 A2 WO 9836636A2
Authority
WO
WIPO (PCT)
Prior art keywords
plant
medium
banana
regeneration
cells
Prior art date
Application number
PCT/EP1998/001125
Other languages
English (en)
Other versions
WO1998036636A3 (fr
Inventor
Hilde Maria Emmy Schoofs
Laszlo Sagi
Bartholomeus Jozefus Panis
Serge Remy
Rony Leon Swennen
Original Assignee
K.U. Leuven Research & Development
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by K.U. Leuven Research & Development filed Critical K.U. Leuven Research & Development
Priority to AU67265/98A priority Critical patent/AU6726598A/en
Publication of WO1998036636A2 publication Critical patent/WO1998036636A2/fr
Publication of WO1998036636A3 publication Critical patent/WO1998036636A3/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
    • C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers

Definitions

  • the present invention relates to a new process for the preparation of regenerable banana embryogenic cell cultures and to their use for long-term storage and for producing optionally transgenic banana plants. Due to the ease of handling and production seed is by far the preferred vehicle for the propagation and cultivation of most agronomic and forest tree species. However, in some instances uniformly homogeneous seed can either not be produced or utilized for a number of agronomical and horticultural crops. For example, ten out of thirty crops with an annual production of between 10 and 450 million metric tonnes are vegetatively propagated. These crops thus require an alternative vehicle that may be regenerated into a complete plant. Large-scale and uniform multiplication of these crops is achieved by means of biotechnology which is based on a range of in vitro culture techniques including clonal propagation of vegetative meristematic tissues and culture of morphogenic or embryogenic cells and protoplasts.
  • Bananas are important food crops in the humid and subhumid tropical regions of the world. With an annual production of over 85 million metric tonnes in 1994 bananas are the most important fruit crops worldwide and make a very significant contribution to food security in developing countries. More than 10% of the world's production with a value over US $ 4 billion enters the international export trade and generates an enormous source of income for Third World countries .
  • declining yields or epidemics due to the spread of virulent diseases and pests such as black sigatoka, fusarium wilt and the banana bunchy top disease and nematodes and banana weevils have led to increasing efforts for effective crop protection by using a lot of pesticides.
  • not all these pathogens can be treated with chemicals and use of great amounts of pesticides is not only a burden on the environment but also leads to the pests becoming resistant to the pesticide .
  • bananas are food crops that are exclusively vegetatively propagated and are characterised by a low degree of genetic variability.
  • Conventional breeding of cultivated bananas also remains difficult due to high sterility levels and polyploidy of most edible clones.
  • only slow progress can be achieved in classical breeding programmes because they must be initiated with poorly characterised and unimproved clones .
  • Plant tissue culture and molecular genetic techniques have the potential to overcome some of the factors limiting traditional approaches to banana improvement and are thus highly desirable both from an economic and an environmental point of view. These procedures do however depend on successful regeneration of plants from cells or protoplasts.
  • Somatic embryogenesis is the particular type of differentiation observed from cells or protoplasts. It is the development of embryo-like structures that appear to recapitulate the typical stages in the embryogenesis of a zygote .
  • Plant regeneration methods based on somatic embryos comprise provision of a cell culture, induction of embryogenesis, culturing of embryos and subsequent regeneration and mass clonal propagation of plants.
  • genetic engineering the addition of a genetic transformation step before plant regeneration is required.
  • protoplasts or embryos cryopreservation methods are useful for long-term storage of the cells.
  • plant regeneration in long-term embryogenic cell suspension cultures as described by Dhed'a et al . (1991) comprises culturing meristematic scalps separated from proliferating shoot -tip cultures in a modified liquid MS medium containing 5 ⁇ M 2 , 4-dichlorophenoxyacetic acid (2,4-D) and 1 ⁇ M zeatin.
  • Other methods are reported on the induction of somatic- embryogenesis from alternative explants such as immature zygotic embryos (Cronauer-Mitra and Krikorian, 1988; Escalant and Teisson, 1989), and immature male inflorescences (Escalant et al . , 1994).
  • the average frequency of somatic embryogenesis was in the range of 12-30% and 2-5% for zygotic embryo explants and male inflorescences, respectively, which was efficient enough for the establishment of embryogenic cell suspensions from both types of explants (Marroquin et al . , 1993; Cote et al . , 1996).
  • a procedure for the induction of cell suspensions from in vitro rhizome and leaf base explants was also described (Novak et al . , 1989) .
  • This method is also characterised by other drawbacks that include low proliferation rates or degeneration of meristematic explants and blackening of the cultures due to the release of polyphenolic substances.
  • high quality scalps can not or at best very rarely can be obtained for initiation of somatic embryogenesis.
  • the method requires prolonged (up to 6 months) culture in liquid medium which increases chances for contamination due to the need for frequent subculturing.
  • a plant regeneration method comprising the provision of a starting material for regeneration and the regeneration of plants from the starting material.
  • the provision of a starting material comprises proliferating in vitro meristems in the presence of cytokinins at a concentration of at least 50 ⁇ M, preferably at least 100 ⁇ M.
  • cytokinins at a concentration of at least 50 ⁇ M, preferably at least 100 ⁇ M.
  • cytokinins at a concentration of at least 50 ⁇ M, preferably at least 100 ⁇ M.
  • the step of the regeneration of plants is initiated by induction of somatic embryogenesis on semi-solid medium on this medium no subcultures are performed.
  • a semi-solid induction medium instead of a liquid one is that it is faster, selective, less time- and space- consuming and that there is a lower risk that it will become contaminated.
  • embryogenic cells are diluted and this requires an additional enrichment step which is time- consuming.
  • the step of induction of embryogenesis is not limited to use together with the method described above for provision of starting materials for regeneration in the form of cell suspensions but may also be used with cultures that are provided by any other method.
  • the meristems are for example shoot tips, but the invention is not restricted to shoot tips.
  • the regeneration of complete plants from optionally transformed cells in culture differs from the regeneration process previously used.
  • the new method is a simplified three-step process in which embryogenic cells are held on semi-solid media resulting in higher plant regeneration frequencies than earlier.
  • the plant regeneration method of the invention may further comprise a previous or intermediate cryopreservation step either prior to or after the provision of the starting material, which cryopreservation step comprises the addition of sucrose to the material to be cryopreserved in a concentration of at least 0.3 M.
  • the material to be cryopreserved may be the starting material, i.e. the proliferating cultures, of the suspension preparation method.
  • sucrose thereto is achieved by preculturing the meristems on media containing high sucrose concentrations.
  • This cryoprotective step is followed by rapid freezing.
  • the material to be cryopreserved can be the end-product of this method, i.e. embryogenic cell suspensions, and the addition of sucrose thereto is achieved by a combined treatment with sucrose and dimethylsulfoxide . These cryoprotected suspensions are subjected to slow freezing.
  • the culturing method of the invention can not only be used to produce embryogenic cell cultures but also somatic embryos and regenerable protoplasts which are suitable for genetic transformation by electroporation and may also be applied to somatic hybridization .
  • the novel or optimized cryopreservation protocols for long-term storage of both proliferating cultures and embryogenic cell suspensions may also be used in combination with other plant regeneration steps than the ones described here or as such. The invention does not exclude these possibilities.
  • the cryopreservation method is also very suitable for general storage of germplasm.
  • either embodiment of the method namely a method with only the new cell suspension step, or only the induction step or both may further comprise a genetic transformation of the cells before plant regeneration.
  • a method with only the new cell suspension step, or only the induction step or both may further comprise a genetic transformation of the cells before plant regeneration.
  • the method of the invention it becomes possible to genetically manipulate cells of all major banana groups in the lab by the addition of the genetic transformation step before plant regeneration.
  • a preferred transformation method to be used in the genetic engineering step in the regeneration method of the invention is described by Sagi et al . in Bio/Technology 13, 481-485 (1995) .
  • Bananas dessert, cooking, highland, beer, ornamental and textile bananas as well as plantains, also commonly described under genus name Musa.
  • Edible bananas are thought to be derived from probably independent inter- and intraspecific hybridization events involving two wild diploid species, M. acuminata and M. balbisiana. The genomic constitution of these two species is designated with AA and BB, respectively. Consequently, depending on how the genomes of these wild species were combined the natural triploid hybrids are characterised by different genomic constitutions. For instance, dessert and highland bananas - mostly AAA, cooking bananas - mostly ABB, plantains - AAB, etc. Approximately 10% of the world banana production enters the international export trade and distributed as dessert banana. By far the most widespread group of dessert bananas is called Cavendish with the commercial .clones named Grande Naine and Williams. Plantains are basic food for approximately a hundred million people because they are rich in carbohydrates and can be consumed fresh, cooked or fried.
  • Cell and tissue culture a complex process under sterile or in vitro conditions that comprises the steps of (i) the production of highly proliferating shoot-tip cultures from which embryogenesis-competent scalps are derived, (ii) in vitro culturing of scalps till the formation of embryogenic cultures, and (iii) subsequent maintenance of cell suspensions.
  • Cytokinins a group of plant growth substances (hormones) that influence plant physiological processes both in nature and in cell and tissue cultures . At the cellular level in the presence of other plant hormones, especially auxins, they promote cell division and determine the direction of cell differentiation. At the tissue level cytokinins induce shoot formation, chloroplast development and leaf expansion and they delay leaf senescence. At the molecular level their action is not well understood. Though a wide range of substances show cytokinin-like activities, two major groups are usually distinguished: (i) N 6 -substituted adenine derivatives and (ii) substituted phenylureas .
  • the first group contains most naturally occurring cytokinins like zeatin, dihydrozeatin, isopentenyladenine and their ribosides or glycosyl conjugates as well as synthetic compounds like kinetin, 6 -benzyladenine and 6- benzyladenosine .
  • Thidiazuron and N,N' -diphenylurea as well as its derivatives are the typical examples of substituted phenylureas.
  • Explant an organ, a tissue, or cells derived from a plant and cultured in vitro for the purpose of starting a plant cell or tissue culture.
  • Initiation the initial cellular proliferation or morphogenic development that eventually results in the establishment of a cell or tissue culture from an explant.
  • Meristem culture the in vitro culture of shoot meristems .
  • Regeneration a morphogenic response to stimuli that results in the production of organs, embryos, or whole plants from cell or tissue cultures.
  • Cryopreservation a method for the long-term storage of cells and tissues, based on the interruption of metabolic activities in cells by the application of ultra- low temperatures, without impairing viability.
  • Genetic transformation the unidirectional transfer and incorporation of foreign purified or recombiriant DNA into the genome of plant cells. For the production of transgenic plants genetic transformation is an additional step before regeneration.
  • Protoplast a plant cell from which the cell wall has been removed.
  • Somatic embryogenesis the process of initiation and development of embryos in vitro from somatic cells and tissues.
  • Embryogenic cells cells which will develop into somatic embryos upon transfer onto the appropriate regeneration medium.
  • Embryogenic culture a plant cell or tissue culture which is capable of forming somatic embryos and regenerating plants via somatic embryogenesis.
  • Embryogenic cultures of bananas consist of a mass of embryogenic cells, early stage somatic embryos and tissues producing such cells and embryos.
  • Somatic embryo formed during the process of somatic embryogenesis or after the transfer of embryogenic cells and tissues to media allowing plant regeneration from embryogenic cells.
  • Early stage somatic embryo morphologically similar to immature zygotic embryos. In this context, such embryos refer to preglobular, globular embryos and embryos in the early stages of cotyledon formation.
  • Plantlet a small plant derived from a germinating somatic embryo.
  • Rooting the emergence of the radicle or root from a somatic embryo.
  • High quality scalp a scalp prepared from shoot -tip cultures with a cauliflower- like aspect and competent for induction of somatic embryogenesis.
  • a proliferation medium consisting of a standard basal plant growth medium including but not restricted to that of Schenk and Hildebrandt (1972) or Murashige and Skoog (1962, MS medium) and supplemented with (i) 0.5 ⁇ M to 2.5 ⁇ M of auxin, preferably indole acetic acid (IAA) and preferably at a concentration of 1 ⁇ M, (ii) 50 ⁇ M to 250 ⁇ M of cytokinin, such as zeatin, dihydrozeatin, isopentenyladenine and their rib ⁇ sides, kinetin and benzyladenosin, preferably benzyladenine (BAP) and preferably at a concentration of 100 ⁇ M, (iii) 2% to 4% (w/v) of a sugar selected from the group sucrose, glucose, maltose, fructose or combinations thereof, preferably sucrose, at a preferable concentration of 3% (w/v), and solidified with 0.2% (w)
  • auxin
  • This proliferation medium is called p4.
  • Meristem cultures are grown at about 24 °C to about 30°C and preferably at 27°C under fluorescent light at about 20 ⁇ einstein/m 2 /s to about 80 ⁇ einstein/m 2 /s of Photosynthetic Active Radiation (PAR) and preferably at 50 ⁇ einstein/m 2 /s of PAR in standard in vitro culture containers, preferably in 150-ml glass test tubes filled with about 18 ml to about 25 ml of medium p4.
  • the photoperiod of cultures is about 12 h to about 24 h and preferably amounts to 16 h. Under these conditions, the cultures are incubated and subcultured each 1 to 3 months.
  • AAA highland bananas Igitsiri, Nakitengwa AAB dessert bananas: Prata, Lady Finger, Silk - AAB plantains: Agbagba/Harton, Three Hand Planty, Bise Egome-1, Dominico Harton
  • This new method that (i) drastically improves in vitro proliferation rates, (ii) results in embryogenesis-competent explants, and (iii) is less susceptible to tissue blackening has been optimised for banana, but is possibly applicable to the order Zingiberales (e.g. Ensete spp., Heliconia spp . , Canna sp . , Strelitzia spp . , Zinqiber spp . ) and other monocotyledonous species like palms and cocoyam.
  • Zingiberales e.g. Ensete spp., Heliconia spp . , Canna sp . , Strelitzia spp . , Zinqiber spp .
  • Scalps are prepared from optimised in vitro banana meristem cultures (see example 1) by taking the upper 2 mm to 6 mm layer of clumps of 2 to 20 proliferating shoot meristems.
  • Scalps of about 1 mm by about 1 mm to about 7 mm by about 7 mm and preferably 3 mm by 3 mm are induced in standard in vitro culture containers (see example 1) filled with semi-solid induction media consisting of a standard basal plant growth medium, preferably half-strength MS medium supplemented with (i) 4 to 20 ⁇ M of auxin, preferably 2,4-D and preferably at a concentration of 5 ⁇ M, (ii) 0 to 2 ⁇ M of cytokinin, preferably zeatin and preferably at a concentration of 1 ⁇ M, (iii) 2% to 6% (w/v) of a sugar selected from the group sucrose, glucose, maltose, fructose or combinations thereof, preferably sucrose, at a prefer
  • This induction medium is abbreviated as ZZ medium.
  • Scalp cultures are grown under the light conditions defined in example 1 and at a temperature between about 18°C and about 30°C, preferably at 27°C and maintained without subculturing until the development of an embryogenic culture (Figure ID) after 5 to 30 weeks of culture depending on the cultivar.
  • the substitution of semi-solid induction medium for liquid medium induces an embryogenic response in a wide range of banana cultivars including but not restricted to Cavendish dessert banana cultivars (Table 1) and reduces the induction time.
  • the maintenance of scalp cultures without subculturing is significantly more effective compared to frequent refreshments and in addition reduced the risk of contamination.
  • the scalp-derived embryogenic cultures contain early-stage somatic embryos and embryogenic cells, which can be regenerated as described in example 4. Any other somatic banana explant suitable for induction of somatic embryogenesis might replace the scalp in the method described above.
  • Scalp-derived embryogenic cultures are induced at a frequency of 15-80% in the following wide range of cultivars :
  • AAA highland bananas Igitsiri, Nakitengwa AAB dessert bananas: Prata, Lady Finger - AAB plantains: Agbagba/Harton, Three Hand Planty, Bise Egome-1, Dominico Harton ABB cooking bananas: Bluggoe, Cardaba, Saba AA edible cultivar: Guyod
  • This example illustrates the proliferation of embryogenic cultures on semi-solid media and the establishment of embryogenic cell suspensions therefrom in liquid medium.
  • embryogenic cultures Prior to transfer to liquid maintenance media, embryogenic cultures are optionally transferred to fresh semi-solid ZZ medium (for composition see example 2) in standard in vitro culture containers preferably Petri dishes or 150-ml glass test tubes filled with 18 ml to 25 ml of ZZ medium.
  • the ZZ medium also allows for a rapid proliferation of embryogenic cell material. Embryogenic banana cultures are subcultured and maintained as such for up to 8 months.
  • Embryogenic cultures induced as described in example 2 or maintained as above, are transferred to liquid maintenance medium which is identical to the induction medium ZZ (example 2) .
  • Per ml of medium 0.02 g to about 0.10 g and preferably 0.04 g fresh weight of embryogenic cell material is incoculated.
  • embryogenic cell suspensions ( Figure 2A) are established with a monthly multiplication rate of 2 to 8 ( Figure 2B) . These embryogenic cell suspensions are regenerated to rooted plants in a time span of 3 months to 8 months (example 4).
  • Embryogenic cell suspensions are maintained in Erlenmeyer flasks on a rotary shaker at about 40 rpm to about 120 rpm and preferably at 70 rpm, and grown under the light and temperature conditions defined in example 1.
  • the established embryogenic cell suspensions are subcultured about every 10 days to about every 30 days and preferably about every 14 days.
  • Scalp-derived regenerable embryogenic cell suspensions are obtained in but are not limited to the following cultivars (Table 1) :
  • Scalp-derived banana embryogenic cell suspensions can be maintained for up to 5 years without loosing their morphogenic potential. These embryogenic cell suspensions are successfully cryopreserved (example 7) and have proven to be most suited to other applications like the isolation of regenerable protoplasts (example 5) and genetic transformation (examples 8 and 9) .
  • Plant regeneration from banana embryogenic cell suspensions is based on the multistep protocol described by Dhed'a et al . (1991) with modifications as described below.
  • Stage 1 The formation of globular to early cotyledonary stage somatic embryos from embryogenic suspension cells
  • a standard plant growth medium preferably the standard MS medium supplemented with 2% to 4% (w/v) of a sugar selected from the group sucrose, glucose, maltose, fructose or combinations thereof, preferably sucrose, at a preferable concentration of 3% (w/v) , 0 mg/1 to 100 mg/1 and preferably 100 mg/1 of myo-inositol , and 0.2% (w/v) Gelrite.
  • This medium which is not supplemented with growth regulators is called the basal regeneration medium.
  • Globular to early cotyledonary stage embryos are continuously formed during a period of 1 to 3 months.
  • the cell suspensions Prior to plating, the cell suspensions are sieved at 500 ⁇ m and washed thoroughly with a liquid hormone-free plant growth medium, preferably the basal regeneration medium as defined above.
  • Per test tube one drop (about 0.05 ml) of a settled cell suspension is added.
  • Petri dishes 0.05 ml to 0.5 ml of a settled cell suspension are used for regeneration.
  • Stage 1 somatic embryos regenerated from cell suspensions or directly derived from the embryogenic cultures are then transferred individually or in small groups to standard in vitro culture containers and preferably 150-ml glass test tubes filled with the basal regeneration medium supplemented with 0 ⁇ M to 10 ⁇ M of cytokinin, preferably BAP and preferably at a concentration of 2 ⁇ M. Addition of cytokinins to the basal regeneration medium enhances subsequent shoot formation.
  • the cultures are incubated under the standard growth conditions (example 1) for 1-3 months.
  • One- to three-cm tall shoots are transferred to standard in vitro culture containers and preferably to 150-ml glass test tubes filled with 18 ml to 25 ml of a rooting medium consisting of the basal regeneration medium supplemented with (i) 1 ⁇ M to 3 ⁇ M of auxin, preferably IAA and preferably at a concentration of 1 ⁇ M, and (ii) 1 ⁇ M to 3 ⁇ M of cytokinin, preferably BAP and preferably at a concentration of 1 ⁇ M.
  • Root formation usually takes place within 1-2 months of culture under the standard growth conditions (example 1) . More than 90% of plants of 10-15 cm height regenerated with this procedure survive the transfer to full ground.
  • the scalp- derived embryogenic cell suspensions may also be applied to large-scale clonal propagation in banana. This could be achieved by mass cultivation in bioreactors .
  • the large number of embryos obtained through somatic embryogenesis can also be used for the production of artificial seeds (Redenbaugh et al . , 1986) by encapsulating them in alginate or agar beads.
  • embryogenic cell suspensions have been the only source of regenerable protoplasts.
  • embryogenic cell suspensions of bananas proved to be suitable for the isolation of protoplast with capacity for plant regeneration (Panis et al . , 1993).
  • regenerable protoplasts are suitable for genetic transformation by electroporation (example 6) and may also be applied to somatic hybridization. Protoplast isolation.
  • embryogenic cells of but not restricted to the cooking banana cultivar Bluggoe are concentrated to a packed cell volume (PCV) between 10% and 30% and preferably 20% (v/v) .
  • PCV packed cell volume
  • One ml of this concentrated suspension is subsequently transferred to 6 ml of enzyme solution containing cellulase Onozuka R-10 from Trichoderma viridae, macerozyme R-10 from Rhizopus spp., and pectinase 5S from Aspergillus niger alone or in combinations at a concentration of 0.1% to 2.0%.
  • the enzyme solution also contains 7 mM CaCl 2 2H 2 0, 0.7 mM NaH 2 P0 4 2H 2 O, 3 mM 2-N-morpholinoethane sulphonic acid and 10% (w/v) mannitol.
  • the pH is adjusted to 5.8 and the solution filter-sterilized. Cells are incubated at 40 rpm on a rotary shaker in the dark for periods ranging between 4 h and 24 h.
  • Optimal protoplast yields using a 2 -week-old suspension are obtained after 24 hours digestion with a combination of cellulase and macerozyme and pectinase each at a concentration of 1% (w/v) and reach as many as 70 million protoplasts per ml PCV of cells .
  • Protoplast purification After the enzyme treatment, the protoplast- containing suspension still contains undigested cell clumps and cell debris. Therefore, the suspension is washed by centrifugation at 66 g for 5 min. with 10 ml MS medium devoid of plant growth regulators but supplemented with 10% (w/v) mannitol. Then, protoplasts are purified either by flotation on 20% to 25% (w/v) sucrose or sieving through a 100- ⁇ m and subsequently through a 25- ⁇ m filter. Purified protoplasts are counted before and after purification to determine purification efficiencies. Cell wall degradation and viability are visualized with a UV fluorescent microscope using Calcofluor white and fluorescein diacetate, respectively. The purification efficiency with the flotation method varies between 15% and 35%, while the sieving method results in only 0-26% loss of protoplasts after purification.
  • Protoplast culture Protoplasts are resuspended at densities ranging between 10 5 and 10 6 protoplasts per ml in half- strength Murashige and Skoog (MS) medium containing 5 ⁇ M 2,4-D and 10% (w/v) mannitol. Three regeneration methods are successfully applied. 1/ Culture on solid medium:
  • 2 ml of the nurse culture is placed which is obtained by mixing 1 ml of a 1-week-old embryogenic cell suspension concentrated to 5% (v/v) PCV with 1 ml MS medium supplemented with 5 ⁇ M 2,4-D, 10% (w/v) mannitol, 200 mg/1 myo-inositol , and 1.6% (w/v) agarose.
  • a black Millipore filter (see above) is placed, and onto the filter 1 ml of protoplasts is gently poured.
  • 3/ Culture on preconditioned medium This method resembles the previous one. However, instead of 1 ml of embryogenic cell suspension, 1 ml of a preconditioned medium is used. The preconditioned medium is prepared by separating the medium from a 1-week-old cell culture with a Whatman filter followed by filter- sterilization.
  • Cryopreservation of proliferating meristems serves two purposes. Firstly, the starting material for induction of embryogenic cell cultures can be stored without the risk of microbial contamination. Secondly, transgenic shoot cultures can be stored safely on a long term. Recently, a cryopreservation method for proliferating meristems has been developed (Panis et al . , 1996) which consists of a meristem preculture step on media containing high sugar concentrations followed by rapid freezing. Post-thaw culture proceeds on semi-solid regeneration medium. In this example, we describe an improved cryopreservation protocol using liquid regeneration media which results in significantly higher post-thaw survival rates.
  • Preculture From proliferating meristem cultures, white meristematic clumps of about 4 mm diameter, each containing at least four apical domes are excised and transferred onto a preculture medium.
  • This medium consists of semi-solid MS medium supplemented with 10 ⁇ M BAP and 1 ⁇ M IAA and enriched with sucrose, glucose or fructose or a combination of these sugars to a final concentration of 0.3 M to 0.6 M, preferably sucrose and preferably at a concentration of 0.4 M. These cultures are kept for about 1 week to about 4 weeks and preferably for about 2 weeks under conditions identical to those for normal meristem culture (example 1) .
  • Thawed explants are transferred to 100-ml Erlenmeyer flasks containing 30 ml liquid MS medium supplemented with 10 ⁇ M BAP, 1 ⁇ M IAA and 3% (w/v) sucrose, and placed on a rotary shaker at about 40 rpm to about 90 rpm and preferably at about 70 rpm.
  • the cultures are placed for 1 week in the dark after which they are placed in the culture room under standard growth conditions.
  • Six weeks after freezing, regrowth is determined under a binocular microscope.
  • the regeneration frequencies of a number of banana cultivars belonging to different genomic groups are listed in Table 3.
  • Recovering clumps are transferred to test tubes containing a semi-solid MS medium supplemented with 1 ⁇ M BAP, 1 ⁇ M IAA and 3% (w/v) sucrose. As soon as rooted plants reach the top of the test tube, they are transplanted in the soil. Table 3
  • cryopreservation During prolonged cell suspension culture there is always a risk of loosing this material due to microbial contamination, loss of regeneration capacity and somaclonal variation. It is therefore important to conserve this valuable material in a secure way.
  • panis et al . (1990) described a freezing method for embryogenic cell suspensions of the cooking banana cultivar Bluggoe. However, generalization of this freezing method towards embryogenic cell suspensions of a large number of banana cultivars can only be obtained if high concentrations of sucrose are added to the cryoprotective medium.
  • the present cryopreservation protocol is developed for established embryogenic cell cultures (example 3). However, scalp- derived embryogenic cultures (example 2) and globular somatic embryos (example 4) may also be subjected to cryopreservation.
  • cryotubes Freezing, storage and thawing.
  • One and a half ml aliquots of the cryoprotected suspensions are transferred to 2 -ml cryotubes and placed in a stirred methanol bath. This methanol bath cools at a rate of l°C/min.
  • the cryotubes are immersed for 3 seconds in liquid nitrogen to initiate ice crystallization. Then, they are further cooled to -40°C. After 30 min. at -40°C, the cryotubes are immersed for storage in liquid nitrogen. Subsequently, cryotubes are rapidly thawed in a beaker filled with sterile water of 40°C for about 1.5 min. until most of the ice is melted.
  • the reinitiated cell suspensions are regenerated in culture tubes containing semi-solid basal regeneration medium following the protocol described in example 4.
  • This cryopreservation method is successful for embryogenic cell suspensions of several cultivars belonging to different genomic groups including but not restricted to the Cavendish group of dessert bananas, the cooking bananas, the plantains, and wild diploid species such as Musa balbisiana. Since these groups represent a significant range of genetic diversity in bananas it is assumed that this cryopreservation method offers wide if not universal applicability in banana.
  • ECSs Embryogenic cell suspensions
  • the ECSs are cultured for at least 1 year prior to use in the experiments and consist of small clusters of isodiametric, cytoplasm-rich cells.
  • the plasmids used for transformation are propagated in the E. coli DH5c. strain.
  • Plasmid DNA prepared by alkaline lysis is purified on Qiagen columns (Diagen GmbH) . DNA concentrations are determined by absorption measurements at 260 nm and by gel electrophoresis.
  • Isolation and purification of protoplasts from embryogenic cell suspensions is done by strictly following the procedure outlined in example 5.
  • Purified protoplasts are resuspended in electroporation buffer at a concentration of 1.25 x 10 6 protoplasts per ml.
  • the electroporation buffer is composed of 70 mM potassium aspartate, 5 mM calcium gluconate, 5 mM 2-N-morpholino- ethane sulphonic acid, and 0.55 M mannitol, the pH is adjusted to 5.8 (Tada et al . , 1990).
  • Protoplasts are counted by using a modified Neubauer haemocytometer . Complete removal of the cell wall is confirmed by Calcofluor white staining while the viability of freshly isolated or electroporated protoplasts is routinely assessed by staining either with fluorescein diacetate or Evans ' Blue .
  • Transformation procedure For transformation of protoplasts by electroporationm is described by Sagi et al . (1994). A 800 ⁇ l aliquot containing 10 6 protoplasts in electroporation buffer is placed into an electroporation cuvette with a 0.4 cm gap. After addition of plasmid DNA to a concentration of 60 ⁇ g/ml, cuvettes are stored on ice for 10 min., then electroporated with a 960 ⁇ F capacitor of a Bio-Rad Gene PulserTM transfection apparatus or of an equivalent at a field strength of 800 V/cm. After electroporation, the cuvettes are placed on ice for 10 min. and then for 10 min. room temperature.
  • Protoplasts are diluted with ZZ medium supplemented with 0.55 M mannitol to a concentration of 10 5 protoplasts per ml and incubated in the dark at 24 °C.
  • the following controls are used: (i) samples electroporated with pUC19 plasmid DNA, (ii) samples electroporated without plasmid DNA, (iii) non-electroporated samples incubated with the plasmid DNA used for electroporation.
  • protoplasts are collected 48 hours after electroporation, resuspended in 50 mM sodium phosphate buffer, pH 7.0 and incubated for periods ranging from overnight up to 10 days at 37°C in the presence of 1 mM X-Gluc (5-bromo-4- chloro-3-indolyl- ⁇ -D-glucuronide) as described by Jefferson (1987) .
  • Protoplast transformation frequency is assessed by counting blue-stained protoplasts and relating this to the total number of protoplasts. Two to four internal repeats are counted and averaged for each treatment in each experiment . On average 10 5 protoplasts are observed for each repeat. The total number of treated protoplasts is determined independently for each repeat. Cultures of E. coli are used in parallel as positive controls for the GUS assay.
  • ECSs Embryogenic cell suspensions
  • proliferating meristems or scalps of various banana cultivars including but not restricted to the Cavendish dessert banana cultivar Williams (Musa spp., AAA group), and those described in example 3 are used for genetic transformation.
  • These ECSs are cultured in ZZ medium (described in example 2) , maintained on a rotary shaker as described in example 3, and subcultured at least once but preferably twice each two weeks. Cells are collected about 3 days to about 6 days after subculture and preferably preferably 4 days after subculture and used for genetic transformation.
  • Proliferating meristems are subcultured on medium p4 as described in example 1 and directly used for transformation. Scalps are prepared for transformation before or during induction of somatic embryogenesis as outlined in example 2. 5
  • Plasmid DNA is isolated from the E. coli DH5 ⁇ strain by standard large-scale purification methods using a combination of alkaline lysis and polyethylene glycol
  • 15 17 is performed as follows. About 2 mg to 4 mg of particles and preferably 3 mg of particles is mixed with about 3 ⁇ g DNA to about 6 ⁇ g DNA, and preferably 5 ⁇ g DNA (at a concentration of about 0.6 ⁇ g/ ⁇ l to about 1.2 ⁇ g/ ⁇ l but preferably at 1.0 ⁇ g/ ⁇ l) and while vigorously
  • particles coated with DNA are resuspended in 50 ⁇ l of 100% (v/v) ethanol and pipetted (8 ⁇ l/shot) onto about 100 ⁇ m to about 1000 ⁇ m plastic or stainless steel meshes to allow for drying (between about 1 min. to about 5 min., preferably for about 2 min.) before particle
  • Particle bombardment is carried out with a home-made helium-driven particle gun constructed according to Sagi et al .
  • target distance about 8 cm to about 14 cm, preferably about 12 cm
  • helium pressure about 6 bar to about 10 bar, preferably about 8 bar
  • vacuum about -0.7 bar to about -0.9 bar, and preferably about -0.8 bar
  • one shots to three shots per target but preferably one shot per target and (v) with or without an about 4 hours to about an overnight osmotic pretreatment on ZZ medium containing 0.2 M sorbitol plus 0.2 M mannitol.
  • Growth kinetics are determined for different ECSs by measuring the fresh weight increase of equal amount of embryogenic cells which are plated on selective ZZ media containing the following concentrations (mg/1) of the selective agents kanamycin, geneticin and hygromycin: 5, 10, 20, 50, 75, and 100. Measurements are carried out at two time points, two and four weeks of culture after the treatment started. All measurements are carried out in at least two replicates with two independent experiments. Selective agents and concentrations are considered to be optimal when minimal or no cell growth is observed after the two or four weeks of culture. Viability of killed or surviving cells is also confirmed by standard staining tests like those with fluorescein diacetate or triphenyltetrazolium chloride.
  • ECSs are cultivated on a solid ZZ medium (described in example 2) without selection for about 7 days to about 14 days, and preferably for about 10 days and then placed on solid or in liquid selective ZZ medium as described in example 3 but supplemented with about 25 mg/1 to about 75 mg/1, and preferably 50 mg/1 geneticin or hygromycin and subcultured each 2 weeks.
  • a solid ZZ medium described in example 2
  • active growing cell aggregates are selected and individually subcultured on solid proliferation medium for 1-2 months with or without selection, then transferred onto non-selective or selective proliferation and regeneration medium as described in example 4.
  • Histochemical and fluorometric GUS assays for transient and stable expression in transformed cells, and plant tissues are performed under standard assay conditions (Jefferson, 1987) .
  • Genomic DNA is extracted for PCR and Southern analysis using a standard CTAB extraction method.
  • a 50 ⁇ l PCR reaction mix contains the primers (1 ⁇ M final concentration each), Taq polymerase (1.0 U) , 125 ⁇ M of each dNTP, 1 x PCR reaction buffer and approximately 50 ng DNA. Reactions are overlaid with 50 ⁇ l of mineral oil. PCR conditions are 94 °C initial melting for 3 min. followed by 30 cycles of 94 °C/l min., 60 °C/l min., 72 °C/2 min., with a 72 °C/7 min. final extension. PCR products and total genomic DNA are separated in 1.2% and 0.8% agarose gel, respectively, . and blotted onto nylon membranes. Digoxigenin-labelled probes are prepared by PCR as described above, and used for hybridization according to standard protocols.
  • A. Embryogenic cell aggregates in a cell suspension of the Cavendish banana cultivar Williams (bar 10 ⁇ m) .

Abstract

La présente invention concerne un procédé de régénération d'une plante, consistant la fourniture d'un matériel de départ destiné à être régénéré ainsi que la régénération de plantes à partir de ce matériel de départ, ce procédé étant caractérisé en ce que la fourniture d'un matériel de départ consiste à combiner une sélection rigoureuse de tissu à méristématisation importante avec des méristèmes proliférant in vitro, en présence de cytokinines dont la concentration est d'au moins 50 νm et, de préférence, d'au moins 100 νm, afin d'obtenir des cellules en suspension. Dans ce procédé, on commence idéalement l'étape de régénération des plantes en induisant une embryogenèse somatique de cellules en suspension sur un milieu semi-solide. On peut également utiliser cette étape d'induction dans d'autres procédés de préparation de suspensions de cellules.
PCT/EP1998/001125 1997-02-21 1998-02-23 Procede de production de cultures cellulaires embryogenes pour la production de bananes transgeniques (de l'espece musa) WO1998036636A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU67265/98A AU6726598A (en) 1997-02-21 1998-02-23 Method for generating embryogenic cell cultures for the production of transgenicbananas (musa spp.)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP97200514A EP0861589A1 (fr) 1997-02-21 1997-02-21 Procédé pour générer des cultures cellulaires embryogéniques destinées à la production de bananes (musa spp.)
EP97200514.4 1997-02-21

Publications (2)

Publication Number Publication Date
WO1998036636A2 true WO1998036636A2 (fr) 1998-08-27
WO1998036636A3 WO1998036636A3 (fr) 1998-12-03

Family

ID=8228039

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1998/001125 WO1998036636A2 (fr) 1997-02-21 1998-02-23 Procede de production de cultures cellulaires embryogenes pour la production de bananes transgeniques (de l'espece musa)

Country Status (5)

Country Link
EP (1) EP0861589A1 (fr)
AU (1) AU6726598A (fr)
BE (1) BE1011836A4 (fr)
CO (1) CO4790178A1 (fr)
WO (1) WO1998036636A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101869076A (zh) * 2010-07-06 2010-10-27 广东省农业科学院作物研究所 一种香蕉吸芽茎尖接种的方法
WO2020014664A1 (fr) * 2018-07-12 2020-01-16 Berkeley Lights, Inc. Le dépistage des protoplastes végétaux pour la recherche de caractéristique de résistance aux maladies
CN111073903A (zh) * 2019-11-15 2020-04-28 青岛农业大学 利用基因枪法和液体培养基抗性筛选的香蕉遗传转化方法
CN113142055A (zh) * 2021-04-29 2021-07-23 广西壮族自治区农业科学院 一种香蕉种质资源离体增殖的保存方法
CN113615575A (zh) * 2021-08-30 2021-11-09 中国热带农业科学院海口实验站 一种香蕉胚挽救的方法

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2007352625B2 (en) * 2007-05-07 2011-12-08 University Of South Carolina Method for micropropagation of monocots based on sustained totipotent cell cultures
US7863046B2 (en) 2007-05-07 2011-01-04 The University Of South Carolina Method for micropropagation of monocots based on sustained totipotent cell cultures
CN106212281B (zh) * 2016-07-28 2017-10-27 广西陆川县乌坭坡珍珠番石榴专业合作社 一种提高香蕉成活率的组织培养方法
CN107232055A (zh) * 2017-06-14 2017-10-10 中国热带农业科学院海口实验站 基于改良培养基的阿希蕉培育方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0400553A1 (fr) * 1989-05-29 1990-12-05 Schweizerische Eidgenossenschaft Eidgenössische Technische Hochschule (Eth) Méthode pour la production de plantes transgéniques
EP0424047A1 (fr) * 1989-10-17 1991-04-24 Pioneer Hi-Bred International, Inc. Méthode de culture de tissus pour la transformation des cellules végétales

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0400553A1 (fr) * 1989-05-29 1990-12-05 Schweizerische Eidgenossenschaft Eidgenössische Technische Hochschule (Eth) Méthode pour la production de plantes transgéniques
EP0424047A1 (fr) * 1989-10-17 1991-04-24 Pioneer Hi-Bred International, Inc. Méthode de culture de tissus pour la transformation des cellules végétales

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE CABA - AN 89:99212 Tropical Agriculture 1988 Vol. 65 No. 4 "Micropropagation of Musa" Mateille, T. XP002078781 *
DATABASE CABA - AN 97:78278 Fruits 1996 Vol 51 No 4 "Mitotic instability in banana varieties BAP concentrations and effects of number of subcultures" Shepherd, K et al. XP002078780 *
DHED'A, D.: "Culture de suspensions cellulaires embryogéniques et régénération en plantules par embryogénèse somatique chez le bananier" TROPICULTURA, vol. 10, no. 4, 1992, pages 152-154, XP002041686 cited in the application *
KRIKORIAN: "Section VI: Tropical and Subtropical Fruits - Chapter 12: Banana" HANDBOOK OF PLANT CELL CULTURE,1984, pages 327-348, XP000618226 *
SAGI, L. ET AL: "Transient gene expression in electroporated banana" PLANT CELL REPORTS, no. 13, 1994, pages 262-266, XP000617868 cited in the application *
TEISSON: "In vitro culture of tropical crops in CIRAD" AGRITROP, vol. 13, no. 2, 1989, pages 1-4, XP002041687 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101869076A (zh) * 2010-07-06 2010-10-27 广东省农业科学院作物研究所 一种香蕉吸芽茎尖接种的方法
WO2020014664A1 (fr) * 2018-07-12 2020-01-16 Berkeley Lights, Inc. Le dépistage des protoplastes végétaux pour la recherche de caractéristique de résistance aux maladies
CN111073903A (zh) * 2019-11-15 2020-04-28 青岛农业大学 利用基因枪法和液体培养基抗性筛选的香蕉遗传转化方法
CN113142055A (zh) * 2021-04-29 2021-07-23 广西壮族自治区农业科学院 一种香蕉种质资源离体增殖的保存方法
CN113615575A (zh) * 2021-08-30 2021-11-09 中国热带农业科学院海口实验站 一种香蕉胚挽救的方法

Also Published As

Publication number Publication date
CO4790178A1 (es) 1999-05-31
EP0861589A1 (fr) 1998-09-02
BE1011836A4 (nl) 2000-02-01
WO1998036636A3 (fr) 1998-12-03
AU6726598A (en) 1998-09-09

Similar Documents

Publication Publication Date Title
Ozias-Akins Plant regeneration from immature embryos of peanut
US7326826B2 (en) Selection of fungal resistant grape somatic embryos
Rout et al. Recent trends in the biotechnology of Chrysanthemum: a critical review
Khawar et al. Effect of thidiazuron on shoot regeneration from different explants of lentil (Lens culinaris Medik.) via organogenesis
Bhagwat et al. Stimulation of in vitro shoot proliferation from nodal explants of cassava by thidiazuron, benzyladenine and gibberellic acid
Gray Somatic embryogenesis in grape
Vijayan et al. Application of tissue culture techniques for propagation and crop improvement in mulberry (Morus spp.)
Debnath Strategies to propagate Vaccinium nuclear stocks for the Canadian berry industry
Johri Experimental embryology of vascular plants
Mori et al. Callus formation and plant regeneration in various Lilium species and cultivars
Corredoira et al. Somatic embryogenesis in chestnut
Flores et al. In vitro culture of grain and vegetable amaranths (Amaranthus spp.)
US6197587B1 (en) Methods and tissue culture media for inducing somatic embryogenesis, agrobacterium-medicated transformation and efficient regeneration of cacao plants
WO1998036636A2 (fr) Procede de production de cultures cellulaires embryogenes pour la production de bananes transgeniques (de l'espece musa)
Nakano et al. Plant regeneration from protoplasts of Gentiana by embedding protoplasts in gellan gum
Kumar et al. Review Article In vitro propagation of kiwifruit
US5312801A (en) Somatic embryogenesis and plant regeneration of cacao
CA1281905C (fr) Regeneration des plantes par organogenese et variation somaclonale chez des plants de glycine
CA2276003A1 (fr) Methode pour la maturation et la culture rapides de plants de ginseng regeneres a partir de cultures d'embryons somatiques
Thomas Advances in mulberry tissue culture
Kunitake et al. Somatic embryogenesis and its application for breeding and micropropagation in asparagus (Asparagus officinalis L.)
Deb et al. Development of a cost effective in vitro regenerative protocol of Cymbidium aloifolium (L.) Sw. using nodal segments as an explants source
Niizeki Haploid, polyploid and aneuploid plants from cultured anthers and calluses in species of Nicotiana and forage crops
Trigiano et al. Somatic embryogenesis in woody legumes
Zhang et al. Improved induction of somatic embryo in watermelon [Citrullus lanatus (Thunb.) Matsum. et Nakai]

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM GW HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM GW HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase in:

Ref country code: JP

Ref document number: 1998536286

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase